DNA vaccines and gene medicines are emerging as an important new class of pharmaceuticals that are derived from bacterial plasmids. For use in humans, plasmid DNA should be essentially free from bacterial genomic DNA and RNA, protein and endotoxin. The hypothesis is that E. coli plasmid DNA can be more efficiently purified by co-expressing RNase and DNase in plasmid producing strains of bacteria. The strategy will be to make hybrid proteins (expressing RNase A and phage T5 DNA exonuclease) and secrete them into the periplasmic space, releasing them at the appropriate time to allow digestion of E. coli RNA and genomic DNA. In Specific Aim I, we will construct recombinant genes for expression of RNase and T5 exonuclease. In Specific Aim II, we will investigate the use of the recombinant enzymes in the purification of plasmids. The overall goal of the proposed Phase I feasibility study is to determine whether such a bacterial strain is a significant advantage in the purification of plasmid DNA. If successful, Phase II studies will include improved constructs for insertion of the genes into the E. coli chromosome; creating the strains needed; development of a third enzyme (protease); and development of an inducible system for introducing the foreign proteins into the cytoplasm during the final hours of fermentation. In Phase Ill, NTC will introduce the improved strains in commercial plasmid production. Success in constructing one or more useful strains of bacteria expressing RNAse, DNAse and/or protease enzymes in a controllable fashion (milestone II) would be a significant development for DNA purification on a commercial scale. Such strains will be rapidly introduced into NTC's large scale DNA manufacturing process, and will also be made available for licensing to other commercial and academic users through the company's technology transfer and licensing program. Currently, experimental gene drugs and DNA vaccines are routinely made on the gram scale, mostly for use in animal safety and efficacy studies. However, the advent of FDA approved commercial products will require scaling up to kilograms, for example, for a widely used vaccine. Judging from the current price of $22,000- $46,000/gram of DNA produced, a less costly, scalable fermentation process is badly needed. The economic benefits and potential market size for the resulting products can be readily appreciated. The ability to use endogenously produced recombinant enzymes would be a significant advantage (in terms of safety and economy) over the use of animal-derived or exogenously added enzymes. The likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.